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61.
Hengxing Lu Jun Liu Tao Feng Zihang Guo Yunjun Yin Fei Gao Gengsheng Cao Xuguang Du Sen Wu 《Genome research》2021,31(5):900
Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.Following the completion of animal genome sequencing projects, rapid and efficient mutagenesis strategies are needed for analyzing gene function and for creating human disease models. Gene trapping is a high-throughput mutagenesis strategy whereby random vector insertion can be achieved across the mouse genome. A typical gene-trap vector contains a promoter-less reporter/selection gene flanked by an upstream splice acceptor (SA) and a downstream poly(A) signal. Upon insertion into an intron of a gene, the vector both inactivates the trapped gene and enables the gene-specific expression of a reporter gene (Gossler et al. 1989; Stanford et al. 2001). To date, gene-trapping approaches have been successfully applied toward large-scale mutagenesis in mouse embryonic stem cells (mESCs) and generation of gene knockout mice (Skarnes et al. 2004). The main drawback of random gene trapping is that gene-trap alleles are not specifically engineered to target genes of interest in advance. Therefore, methods to streamline the introduction of predesigned, site-specific modifications into the genome by homologous recombination would represent a significant technological advance. Previously, a hybrid approach combining gene targeting and gene trapping (targeted trapping) enabled mutation of expressed genes in mESCs with high efficiency, using a gene-trap construct flanked by homologous sequences of the target locus (Friedel et al. 2005). Also, homologous recombination is commonly used for creating conditional alleles, which is essential to avoid embryonic lethality and to study the stage- and tissue-specific functions of genes (Branda and Dymecki 2004). However, both standard gene trapping and targeted trapping are only suitable for genes expressed in embryonic stem (ES) cells. Furthermore, construction of targeting donor vectors with homology arms is labor intensive and costly, and the low efficiency of homologous recombination is also a rate-limiting step for gene targeting in mammalian genomes.Recently, by taking advantage of precise genomic double-strand breaks (DSBs) created by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system (Ran et al. 2013; Doudna and Charpentier 2014; Hsu et al. 2014), homology-directed repair (HDR) efficiency was substantially enhanced (Porteus and Carroll 2005), and even donors with short homology arms (Orlando et al. 2010) or single-stranded DNA oligonucleotides (Chen et al. 2011; Quadros et al. 2017) were found to be compatible with site-specific integration. However, each targeting donor for HDR still needs to be customized with gene-specific homology sequences. Because of the lack of ES cells for certain animals such as pigs, sheep, and cattle, the genome must be edited either in a zygote embryo or in a somatic cell for somatic cell nuclear transfer (SCNT) (Reddy et al. 2020). It is still not feasible to achieve large-scale insertional mutagenesis including conditional knockouts in these important species with random gene trapping or HDR-based methods. Also, the problem of genetic mosaicism in embryo editing remains unresolved (Mehravar et al. 2019), prompting a need for technological advances to accelerate genetic modification in somatic cells.Alternatively, the generally more efficient nonhomologous end joining (NHEJ) pathway has been exploited for site-specific insertion of exogenous DNA by simultaneous cleavage of both donor plasmid and genome using programmable nucleases (Cristea et al. 2013; Maresca et al. 2013; Brown et al. 2016; Suzuki et al. 2016; Sawatsubashi et al. 2018). In contrast to HDR-based strategies, NHEJ-mediated insertions do not require gene-specific homology arms, enabling diverse sites to be targeted with a universal donor vector. Therefore, we speculated that a gene-trap cassette could be inserted into a specific locus easily through this mechanism in any cell type.Here, by combining NHEJ-mediated knock-in and gene trapping, we developed a strategy for targeted mutagenesis, especially in somatic cells with low HDR activity, referred to as HIT-trapping. By using a universal donor, this strategy allows us to (1) create null alleles, (2) produce a fluorescent reporter signal that could potentially allow cells with null alleles to be identified very quickly, and (3) produce reversible and conditional alleles that would be very helpful to have in most animal models but are often cumbersome to create. 相似文献
62.
High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes 总被引:5,自引:0,他引:5
Shuber AP; Michalowsky LA; Nass GS; Skoletsky J; Hire LM; Kotsopoulos SK; Phipps MF; Barberio DM; Klinger KW 《Human molecular genetics》1997,6(3):337-347
As more mutations are identified in genes of known sequence, there is a
crucial need in the areas of medical genetics and genome analysis for
rapid, accurate and cost-effective methods of mutation detection. We have
developed a multiplex allele-specific diagnostic assay (MASDA) for analysis
of large numbers of samples (> 500) simultaneously for a large number of
known mutations (> 100) in a single assay. MASDA utilizes
oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA
samples are immobilized on a solid support and a single hybridization is
performed with a pool of allele-specific oligonucleotide (ASO) probes. Any
probes complementary to specific mutations present in a given sample are in
effect affinity purified from the pool by the target DNA. Sequence-specific
band patterns (fingerprints), generated by chemical or enzymatic sequencing
of the bound ASO(s), easily identify the specific mutation(s). Using this
design, in a single diagnostic assay, we tested samples for 66 cystic
fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell
anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations,
four mutations in Canavan disease, four mutations in Fanconi anemia, and
five mutations in BRCA1. Each mutation was correctly identified. Finally,
in a blinded study of 106 of these mutations in > 500 patients, all
mutations were properly identified. There were no false positives or false
negatives. The MASDA assay is capable of detecting point mutations as well
as small insertion or deletion mutations. This technology is amenable to
automation and is suitable for immediate utilization for high-throughput
genetic diagnostics in clinical and research laboratories.
相似文献
63.
The t(X;1)(p11.2;q21.2) translocation in papillary renal cell carcinoma fuses a novel gene PRCC to the TFE3 transcription factor gene 总被引:4,自引:2,他引:4
64.
65.
Apoptotic rate in peripheral T-cell lymphomas. A study using a tissue microarray with validation on full tissue sections 总被引:8,自引:0,他引:8
Rassidakis GZ Jones D Thomaides A Sen F Lai R Cabanillas F McDonnell TJ Medeiros LJ 《American journal of clinical pathology》2002,118(3):328-334
Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas with a wide spectrum of clinicopathologic features, and apoptosis mechanisms may have a role in lymphomagenesis. We assessed apoptotic rate (AR) in 112 PTCLs using a tissue microarray developed in our laboratory and a modified terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The mean AR was 1.47% +/- 1.38% for the entire group of PTCLs (range, 0.06%-5.15%), and AR varied significantly among different tumor types. In mycosis fungoides, the mean AR was 0.74%; angioimmunoblastic T-cell lymphoma, 1.02%; PTCL, not otherwise specified, 1.38%; cutaneous anaplastic large cell lymphoma (ALCL), 1.41%; anaplastic lymphoma kinase protein (ALK)-negative ALCL, 1.43%; extranodal natural killer/T-cell lymphoma of nasal type, 2.04%; ALK-positive ALCL, 2.95%; and enteropathy-type T-cell lymphoma, 3.06%. Mean AR was higher in PTCL with large cell vs small/medium cell morphologic features (1.66% +/- 1.1% vs 0.99% +/- 1.0%). In a subset of 33 PTCLs, the tissue microarray results comparedfavorably with those obtained in full tissue sections. We conclude that the highest ARs in PTCLs are found in enteropathy-type T-cell lymphoma and ALK-positive ALCL, and that AR can be assessed reliably by using a tissue microarray. 相似文献
66.
67.
Role of MyD88 in diminished tumor necrosis factor alpha production by newborn mononuclear cells in response to lipopolysaccharide 总被引:11,自引:0,他引:11 下载免费PDF全文
Yan SR Qing G Byers DM Stadnyk AW Al-Hertani W Bortolussi R 《Infection and immunity》2004,72(3):1223-1229
Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes. 相似文献
68.
69.
Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection 总被引:22,自引:0,他引:22
P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm. 相似文献
70.
Mahadevaiah SK; Odorisio T; Elliott DJ; Rattigan A; Szot M; Laval SH; Washburn LL; McCarrey JR; Cattanach BM; Lovell-Badge R; Burgoyne PS 《Human molecular genetics》1998,7(4):715-727
An RNA-binding motif (RBM) gene family has been identified on the human Y
chromosome that maps to the same deletion interval as the 'azoospermia
factor' (AZF). We have identified the homologous gene family (Rbm) on the
mouse Y with a view to investigating the proposal that this gene family
plays a role in spermatogenesis. At least 25 and probably >50 copies of
Rbm are present on the mouse Y chromosome short arm located between Sry and
the centromere. As in the human, a role in spermatogenesis is indicated by
a germ cell-specific pattern of expression in the testis, but there are
distinct differences in the pattern of expression between the two species.
Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are
female due to a position effect resulting in non-expression of Sry ;
sex-reversing such mice with an Sry transgene produces males with a high
incidence of abnormal sperm, making this the third deletion interval on the
mouse Y that affects some aspect of spermatogenesis. Most of the copies of
Rbm map to this deletion interval, and the Yd1males have markedly reduced
Rbm expression, suggesting that RBM deficiency may be responsible for, or
contribute to, the abnormal sperm development. In man, deletion of the
functional copies of RBM is associated with meiotic arrest rather than
sperm anomalies; however, the different effects of deletion are consistent
with the differences in expression between the two species.
相似文献